Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34+ cells

The ability of ex vivo expansion to increase the long-term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this point in a baboon model based on autologous transplantation of serial limiting doses of non-manipulated or ex vivo-expanded mobilized CD34+ cells and determined the threshold doses of non-manipulated and expanded cells which supported long-term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3-ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties [semisolid assays, long-term culture-initiating cells (LTC-IC) and non-obese diabetic severe combined immunodeficient reconstituting cells (SRC)]. Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12.3-fold, 16.9-fold, 3.7-fold, 3.5-fold and 2.2-fold increases in CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte CFU (CFU-MK), LTC-IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long-term engraftment was 0.5 x 10(6)/kg CD34+ cells in the control group and 0.3 x 10(6)/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long-term engraftment (r > 0.80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long-term reconstituting cells.     

CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages

The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin(-)c-Kit(+) long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin(-)c-Kit(+)CD34(-) marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin(-)c-Kit(+) CD34(high) HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin(-)c-Kit(+)CD34(-) cells after 16 weeks, indicating that adult Lin(-)c-Kit(+)CD34(-) HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.     

5-Azacytidine supports the long-term repopulating activity of cord blood CD34(+) cells

DNA methylation plays important roles in a wide range of biological phenomena, especially in the embryonic development and tumorigenesis. However, correlations between differentiation and DNA methylation have not been clarified well in each differentiation system. In this study, we focused our attention on regulatory roles of DNA methylation in normal hematopoietic differentiation using a demethylating reagent, 5-azacytidine (5-AzaC). As a source of hematopoietic progenitor cells, we used CD34(+) cells prepared from human umbilical cord blood and examined the effects of 5-AzaC on the colony-forming activity and the long-term culture-initiating (LTC-IC) activity of these cells. 5-AzaC treatment increased LTC-IC frequency 1.57- to 2.50-fold as compared to the nontreated control. In parallel to this, immunoblotting analysis showed that the intensity of overall DNA methylation decreased after 5-AzaC treatment. These results indicated the involvement of DNA methylation and demethylation in controlling immaturity of hematopoietic progenitor cells and the usefulness of 5-AzaC for regulating this immaturity.     

Reversible cell surface expression of CD38 on CD34-positive human hematopoietic repopulating cells

OBJECTIVE: Although increased expression of CD38 on the surface of human CD34(+) cells is associated with differentiation, we reported recently that both lineage-negative (Lin(-)) CD34(+)CD38(-) and Lin(-)CD34(+)CD38(lo) fractions of cord blood contain primitive severe combined immunodeficient (SCID)-repopulating cells (SRC). Thus, it is important to determine if a hierarchical relationship exists between the SRC from these two populations or if CD38 is reversibly expressed. MATERIALS AND METHODS: To determine if SRC from the CD34(+)CD38(-) and CD34(+)CD38(lo) cell fractions could generate SRC of the same and/or alternate CD38 expression, cells from primary nonobese diabetic/SCID mice transplanted with CD34(+)CD38(-) cells were resorted into both CD34(+)CD38(-) and CD34(+)CD38(lo) fractions and injected into separate secondary recipients, which were evaluated for human cell engraftment 7 to 10 weeks later. As primary mice transplanted with CD34(+)CD38(lo) cells also contained cells of both immunophenotype, these cells were also resorted and transplanted into separate secondary recipients. The cell-cycle status of various CD34(+) SRC fractions were evaluated using Hoechst 33342 and Pyronin Y staining in order to determine if CD38 expression was coordinated with divisional activation. RESULTS: Each cell fraction obtained from primary recipients was able to reconstitute secondary mice, indicating that CD38 expression reversibly oscillates between negative and low levels on CD34(+) repopulating cells. CD38 expression on repopulating cells correlated with a transition between the G(0) and G(1) phases of the cell cycle. CONCLUSION: CD38 is reversibly expressed on CD34(+) SRC between negative and low levels and corresponds to a change in the cell-cycle state. These observations establish a foundation to uncover the molecular program of stem cell regulation and underscore the importance of functional assessments when isolating and characterizing human hematopoietic stem cells.     

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.     

Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony- stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady- state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = - .86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = - .70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.     

Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.     

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

CD34+ selected stem cell boosts can improve poor graft function after paediatric allogeneic stem cell transplantation

Poor graft function (PGF) is a severe complication of haematopoietic stem cell transplantation (HSCT) and administration of donor stem cell boosts (SCBs) represents a therapeutic option. We report 50 paediatric patients with PGF who received 61 boosts with CD34⁺ selected peripheral blood stem cells (PBSC) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. Within 8 weeks, a significant increase in median neutrophil counts (0·6 vs. 1·516 × 10⁹/l, P < 0·05) and a decrease in red blood cell and platelet transfusion requirement (median frequencies 1 and 7 vs. 0, P < 0·0001 and <0·001), were observed, and 78·8% of patients resolved one or two of their cytopenias. 36·5% had a complete haematological response. Median lymphocyte counts for CD3⁺, CD3⁺CD4⁺, CD19⁺ and CD56⁺ increased 8·3‐, 14·2‐, 22.‐ and 1·6‐fold. The rate of de novo acute graft‐versus‐host disease (GvHD) grade I–III was only 6% and resolved completely. No GvHD grade IV or chronic GvHD occurred. Patients who responded to SCB displayed a trend toward better overall survival (OS) (P = 0·07). Thus, administration of CD34⁺ selected SCBs from alternative donors is safe and effective. Further studies are warranted to clarify the impact on immune reconstitution and survival.     

Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90- hematopoietic progenitors

To further characterize hematopoietic "replicative stress" induced by bone marrow transplantation (BMT), the cell-cycle status of CD90+/- subsets of marrow CD34+ cells obtained 2 to 6 months after transplantation from 11 fully chimeric recipients was examined. Cycling profiles, derived by flow cytometry after staining with Hoechst 33342 and pyronin Y, were compared with those of 14 healthy marrow donors. Primitive CD34+CD90+ cells represented a smaller proportion of CD34+ cells in recipients (10% +/- 4% versus 19.6% +/- 5.3% in donors; P <.0001) and were more mitotically active, with the proportion of cells in S/G2/M nearly 4-fold higher than in donors (15.6% +/- 3% and 4.4% +/- 1.6%, respectively; P <.0001). By comparison, there was a modest increase in the proportion of CD34+CD90- progenitors in S/G2/M after BMT (10.9% +/- 1% vs 9.6% +/- 2% in donors; P =.04). Replicative stress after BMT is borne predominantly by cells in a diminished CD34+CD90+ population.     

Myeloperoxidase expression in CD34+ normal human hematopoietic cells

Bone marrow (BM), adult peripheral blood (aPB), and umbilical cord blood (CB) samples contain small proportions of CD34+ cells that include virtually all hematopoietic progenitor cells. Myeloperoxidase (MPO) is considered to be selectively expressed in cells committed to granulomonocytic differentiation. Using flow cytometry and an antibody against MPO, we studied at which stage of normal hematopoietic differentiation CD34+ cells being to express MPO. We consistently observed a characteristic MPO/CD34 staining pattern and found that 35% +/- 9% of CD34+ BM cells express MPO. The MPO+ CD34+ subset and the CD33+ CD34+ subset were of similar size and overlapped considerably. MPO+ CD34+ cells expressed high levels of HLA-D molecules, were weakly CD71/transferrin receptor positive to negative, were CD45RA+ and lacked the CD45RO isoform of the leukocyte common antigen. Additionally, MPO+ CD34+ cells were on average larger in size than MPO- CD34+ cells. Virtually identical phenotypic features have previously been described for in vitro colony-forming granulomonocytic progenitor cells. In vitro clonogenic assays performed with MPO-enriched and MPO-depleted fractions of CD34+ BM cells performed by us also suggest, but do not formally prove, that at least a portion of MPO+ CD34+ cells have in vitro cluster (10 to 50 cells/colony) or colony-forming unit granulocyte-macrophage (> or = 50 cells/colony) forming capacity. CD34+ cells from CB and aPB resembled CD34+ BM cells in that considerable proportions of them coexpressed CD33. However, in contrast to BM, CD34+ cells from CB and aPB samples lacked significant MPO expression and, in line with this, the majority of them (CB, 59% +/- 7%; aPB, 66% +/- 5%) coexpressed CD45RO.     

Combined transplantation of allogeneic bone marrow and CD34+ blood cells

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA- identical and MLC- sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti- CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immune reconstitution following transplantation of autologous peripheral CD34+ cells.

The recovery of lymphocyte count, CD4+ and CD8+ T-cell subsets, natural killer (NK) cells and CD19+ B cells has been evaluated during the first 4 months after the infusion of autologous CD34+ peripheral blood progenitor cells (PBPC; group A; 33 patients) or autologous unselected PBPC (group B; 36 patients) for hematological malignancies. Lymphocyte count promptly recovered in both patient cohorts, although the repopulation of CD3+ T cells occurred more rapidly in group B compared with group A. The count of CD4+ T lymphocytes remained <200/microl during the study period in patients transplanted with CD34+ PBPC, being significantly lower compared with group B (p = 0.0019 and p = 0.0035 on days 30 and 60, respectively). CD8+ T cells rapidly increased both in group A and B and CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T cells displayed an activated phenotype in both groups of patients, coexpressing the HLA-DR antigen throughout the study period. No differences in the repopulation kinetics of NK cells and CD19+ B cells were observed. Further investigations are encouraged to characterize T cell competence following transplantation of CD34+ PBPC.     

自体纯化CD+34细胞移植治疗系统性红斑狼疮的免疫重建研究

目的 探讨自体外周血来源的纯化CD+34细胞移植前后系统性红斑狼疮(SLE)患者免疫学指标的变化,并分析其与SLE发病机制及预后的关系.方法 用流式细胞术、酶联免疫吸附等方法动态监测18例SLE患者移植前及移植后1、3、6、12个月时淋巴细胞亚群、补体C3、补体C4、补体CH50免疫球蛋白等免疫学指标的变化.结果 移植后早期各T细胞亚群均明显减低,除CD45RO+CD+4细胞外,其余各T细胞亚群均于移植后1-3个月开始逐渐回升;补体C3、补体C4、补体CH50于移植后1个月开始显著回升;移植后1年内无复发病例;2例患者移植1年后复发,与持续缓解患者相比,其移植前后各阶段的补体C4水平、移植后3个月及1年的补体CH50水平、移植后6个月的CD45RA+CD+8细胞百分比均明显低于持续缓解患者,移植后1个月的CD45RO+CD+4细胞百分比明显高于持续缓解患者.结论 自体外周血来源的纯化CD+24细胞移植是治疗SLE的有效方法之一,移植前后免疫学指标的变化对分析SLE的发病机制具有重要意义,并可作为判断SLE疗效的预测指标之一.     

Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.     

Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...